Immune responses in vitro. XI. Suppression of primary IgM and IgG plaque-forming cell responses in vitro by alloantisera against leukocyte alloantigens

The effects of alloantisera against leukocyte alloantigens on plaque-forming cell (PFC) responses to sheep erythrocytes and the terpolymer of L-glutamic acid⁶⁰-L-alanine³⁰-L-tyrosine¹⁰ (GAT) by mouse spleen cells in vitro have been investigated. Polyspecific antibodies against both H-2 and non-H-2 alloantigens on responding spleen cells suppressed both IgM and IgG PFC responses; antisera against alloantigens coded for by the K and I regions, but not the D region, of the H-2 complex also effectively suppressed PFC responses. The suppression was not due to cytotoxicity to the spleen cells or anti-immunoglobulin activity in the sera and was directly related to the amount of antiserum added to the cultures. The suppression was specific for spleen cells against which the alloantiserum was directed. The alloantisera suppressed responses most effectively when present during the first 24 h of incubation, and although not rendering lymphoid cells incapable of developing PFC responses after removal of noncell-bound antibody, did act by interfering with successful initiation of the PFC response. The alloantisera suppressed both IgM and IgG PFC responses when directed against alloantigens only on macrophages, but selectively suppressed IgG responses when directed against alloantigens only on lymphoid cells. The alloantisera did not interfere with the ability of macrophages to bind GAT or to support the viability of the lymphoid cells, but did interfere with the ability of macrophage-associated antigen to effectively stimulate antibody responses by the lymphoid cells. Possible mechanisms for the effects of alloantisera on macrophages and the selective suppression of IgG responses when the antisera are directed against alloantigens on lymphoid cells are discussed with reference to our current understanding of genetic restrictions governing cell interactions in the development of antibody responses in mice.     

Genetic control of immune responses in vitro. I. Development of primary and secondary plaque-forming cell responses to the random terpolymer 1-glutamic acid 60-1-alanine30-1-tyrosine10 (GAT) by mouse spleen cells in vitro

In vivo, the antibody response in mice to the random terpolymer L-glutamic acid⁵⁰-L-alanine³⁰-L-tyrosine¹⁰ (GAT) is controlled by a histocompatibility-linked immune response gene(s). We have studied antibody responses by spleen cells from responder and nonresponder mice to GAT and GAT complexed to methylated bovine serum albumin (GAT-MBSA) in vitro. Cells producing antibodies specific for GAT were enumerated in a modified Jerne plaque assay using GAT coupled to sheep erythrocytes as indicator cells. Soluble GAT stimulated development of IgG GAT-specific plaque-forming cell (PFC) responses in cultures of spleen cells from responder mice, C57Bl/6 (H-2^b), F₁ (C57 x SJL) (H-2^b/s), and A/J (H-2^a). Soluble GAT did not stimulate development of GAT-specific PFC responses in cultures of spleen cells from nonresponder mice, SJL (H-2^s), B10.S (H-2^s), and A.SW (H-2^s). GAT-MBSA stimulated development of IgG GAT-specific PFC responses in cultures of spleen cells from both responder and nonresponder strains of mice. These data correlate precisely with data obtained by measuring the in vivo responses of responder and nonresponder strains of mice to GAT and GAT-MBSA by serological techniques. Therefore, this in vitro system can effectively be used as a model to study the cellular events regulated by histocompatibility-linked immune response genes.     

Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. IX. Characterization of Idiotype-specific effector-phase suppressor cells on plaque-forming cell responses in vitro

The ability of T suppressor cells, induced by the intravenous injection of 4-hydroxy-3-nitrophenyl acetyl (NP)-modified syngeneic spleen cells, to affect an ongoing B cell response was studied in vitro. It was found that the expression of NPb idiotype-positive B cells could be selectively inhibited by the addition of antigen-induced suppressor cells in the last 24 h of the in vitro culture. This effector-phase suppression of B cell responses was antigen specific and mediated by an Lyt 1-, Lyt 2+, idiotype-binding, T cell population whose suppressive function was restricted by genes linked to the Igh locus.     

Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. VII. Idiotype-specific suppression of plaque-forming cell responses

The ability of suppressor cells induced by the intravenous administration of 4-hydro-3-nitrophenyl acetyl (NP)-modified syngeneic cells to reduce an idiotypic B cell response was studied in both an in vivo and an in vitro system. Idiotype-positive B cells were assayed by the ability of guinea pig anti-idiotypic antiserum to specifically inhibit idiotype-positive plaque formation. It was found that up to 57% of the PFC response in vivo and 100% of the PFC response in vitro was inhibitable with antiidiotypic antiserum. The expression of these idiotype-positive B cells could be suppressed by the transfer of spleen cells form mice treated 7 d previously with NP coupled syngeneic cels. T cells are both required and sufficient for the transfer of idiotype specific suppression. The induction of these idiotype-specific T suppressor cells directly with antigen suggests that recognition of unique determinants on cell surfaces is important for regulation of lymphoid cell interactions. The role of idiotype-specific suppressor cells in the network of lymphoid interactions is discussed.     

Cellular and genetic control of antibody responses in vitro. III. Immune response gene regulation of accessory cell function

The possibility was investigated that Ir genes regulate the function of cells other than T or B cells in the primary IgM responses to the synthetic antigens trinitrophenylated poly-L-(Tyr,Glu)-poly-D,L-Ala--poly-L-Lys [TNP-(T,G)-A--L]and trinitrophenylated poly-,-(His,Glu)-poly-D, L-Ala--poly-L-Lys [TNP-(H,G)-A--L]. The primary responses of (B10 × B10.A)F(1) spleen cells to both antigens were abrogated by Sephadex G-10 passage, and restored by the addition of spleen adherent cells. The cell type in the spleen adherent cell population active in reconstituting the responses to TNP-(T,G)-A--L and TNP-(H,G)-A--L was a non-T, non-B, radiation-resistant, glass-adherent spleen cell. The responses of Sephadex G-10-passed (responder x nonresponder)F(1) spleen cells to TNP-(T,G)-A--L or TNP-(H,G)-A--L were reconstituted by spleen adherent cells from only responder strains. Spleen adherent cells from F(1) mice reconstituted the responses to both antigens. Spleen adherent cells from each of the strains tested reconstituted the non- Ir gene-controlled response to a third antigen, TNP-keyhole limpet hemocyanin. The inability of spleen adherent cells from nonresponder strains to reconstitute the responses to either TNP-(T,G)-A--L or TNP-(H,G)-A--L was not a result of active suppression induced by the presence of nonresponder adherent cells, since a mixture of responder and nonresponder spleen adherent cells reconstituted the responses to both antigens. The use of spleen adherent cells from recombinant strains demonstrated that the autosomal dominant genes controlling the ability of spleen adherent cells to function as accessory cells in the responses to TNP-(T,G)-A--L and TNP-(H,G)-A--L are located in the K or I-A regions of the responder H-2 complex, the same region(s) of H-2 as the Ir genes controlling overall in vitro and in vivo responsiveness to these antigens.     

Oligomeric IgA: the major component of the in vitro primary response of mouse spleen fragments

The primary antibody response elicited from mouse spleen explants by conjugates of the 3-nitro-5-iodo-4-hydroxyphenylacetic acid (NIP) hapten consisted mostly of the IgA class. Poly-L-lysine, pneumococcal polysaccharide Type SIII, keyhole limpet hemocyanin, and sheep erythrocytes were effective carriers in this system, whereas chicken globulin was not. The anti-NIP response against all of the immunogenic conjugates was detectable in culture media 4 days after explantation and immunization, and reached peak titers by 8–10 days. IgA was identified by sucrose gradient velocity centrifugation in conjunction with the use of a class-specific antiserum. The media collected at 4 days contained low titers of IgM antibody, whereas the peak response at 8 days consisted almost entirely of IgA. The primary response IgA secreted by the spleen fragments was characterized as polymeric by its sedimentation rate through a sucrose gradient, and as polyvalent by its drastically greater avidity for NIP₁₄BSA than for free NIP-aminocaproic acid. Its haptenated phage-inactivating activity was abolished by treatment with 0.1 M 2-mercaptoethanol. These experiments indicate that precursor cells existing in the spleen before primary immunization can give rise to production of polymeric IgA.     

Depressant effect of ambroxol on stimulated functional responses and cell death in rat alveolar macrophages exposed to silica in vitro.

The present study examined the effect of ambroxol on free radical production, granule enzyme release, and cell death in silica-activated rat alveolar macrophages. The action of ambroxol was assayed by measuring changes in the activities of protein kinase C (PKC) and tyrosine kinase (PTK) and in the intracellular calcium level. Ambroxol attenuated the production of superoxide, hydrogen peroxide, and nitric oxide and the release of acid phosphatase and lysozyme in macrophages activated by silica. Staurosporine, genistein, EGTA, and trifluoperazine inhibited the silica-induced free radical production and granule enzyme release. Silica induced the increase in PKC and PTK activities and the elevation of intracellular calcium level in macrophages, which was decreased by ambroxol. Silica induced a cell death and increased the caspase-3 activity in macrophages in a concentration-dependent manner. Ambroxol decreased the silica-induced cell viability loss in macrophages. The results show that ambroxol decreases the stimulated responses and cell death in rat alveolar macrophages exposed to silica, which may be accomplished by inhibition of activation processes, protein kinases, and calcium transport. The inhibitory effect of ambroxol on silica-induced cell death appears to provide the protective effect on pulmonary tissues against the toxic action of silica.     

Adenovirus fiber protein produces synthesis of interferon in mouse spleen and macrophage cultures

The possibility of complementation between herpesvirus papio (HVP) and Epstein-Barr virus (EBV) was investigated. Strain 594S-F9 of HVP, unlike strain P3HR-1 of EBV, is not capable of inducing virus antigen synthesis in the EBV genome-carrying, nonproducer lymphoma cell line Raji. The effects of dual infection with these viruses were studied. With untreated viruses, the percentage of cells positive for viral antigens was equal to or slightly less than that in cultures infected with P3HR-1 virus only. However, if UV-irradiated P3HR-1 virus was employed in the dual infection, the relative number of virus antigen-positive cells was enhanced over cultures infected with P3HR-1 virus alone. These results suggest functional complementation between EBV and HVP.     

Antigens of human trophoblast. Effects of heterologous anti-trophoblast sera on lymphocyte responses in vitro

This report describes the inhibition of human mixed lymphocyte culture (MLC) reactions by rabbit antisera to intact and detergent solubilized, fractionated, human trophoblast membranes. Heat-inactivated antisera were passed through solid-phase immunoabsorption columns of normal human serum and extensively absorbed with human erythrocytes, lymphocytes and liver powder. Immunohistological experiments with these absorbed antisera showed that they reacted brilliantly with syncytiotrophoblast in cryostat sections of human but not baboon or monkey placentae, and not with other normal adult tissues including peripheral blood lymphocytes (PBL). Addition of these antisera to MLC reactions produced significant and reproducible suppression of responses without affecting cell viability. Absorption studies demonstrated complete removal of MLC inhibition and trophoblast membranes but not with PBL or suspensions of HEp-2 cells. Timed experiments showed that optimal inhibition occurred when the antisera were added between 2 and 6 h after culture initiation, and that little suppression was achieved after 18 h. Lymphocytes harvested from MLC reactions after 2 h showed that 3--5% of the cells reacted with PBL/liver-absorbed anti-trophoblast sera, and that unstimulated PBL were negative. Cultures of subhuman primate lymphocytes in the presence of heterologous antisera to human trophoblast membranes showed total inhibition of rhesus:human and human:rhesus MLC, and no suppression of baboon:human or human:baboon reactions, whereas human lymphocytes responded in an exagerated manner when stimulated by baboon cells. Modulated MLC responses to human, rhesus, or baboon lymphocytes, in the presence of anti-trophoblast sera indicate that the antisera recognize trophoblast cross-reactive lymphocytes antigens. We propose that these antigens are reaction products of cell-cell interactions, and that the nature of the antigens is determined by the specificity of the recognition signals which initiate the reaction.     

小鼠脾脏树突状细胞的体外扩增与培养

背景:脾脏来源的树突状细胞诱导周期长,产量相对较少,相关报道亦较少.目的:建立简便的体外扩增小鼠脾脏树突状细胞的方法.设计、时间及地点:细胞形态学观察实验,于2007-02/09在华北石油总医院中心实验室完成.材料:健康BALB/C小鼠、C57小鼠及粒细胞-巨噬细胞集落刺激因子、白细胞介素4.方法:无菌手术取C57小鼠脾脏,用1 mL注射器抽取Hanks液,刺入脾脏并反复冲洗,获取单个核细胞,用0.1 g/L粒-单核细胞集落刺激因了、白细胞介素4的H-DMEM培养,8 d后即得成熟的树突状细胞.同时无菌取BABIJC小鼠脾脏制备单细胞悬液,收集非黏附细胞调整密度后作为反应细胞.再将两种细胞混合培养.主要观察指标:成熟树突状细胞的形态学观察,并对其表面CD11c,CD86及MHC-Ⅱ类分子进行检测.并计算两种细胞混合培养后的吸光度值.结果:体外诱导培养8 d后获得大量成熟的树突状细胞,细胞表面具有典型的树枝状突起,高表达树突状细胞相对特异性表面分子CD11c(78.46%)、CD86(87.24%)及MHC-Ⅱ(92.65%),同时具有较强的刺激同种异基因混合淋巴细胞增殖的能力.结论:联合应用粒细胞-巨噬细胞集落刺激因子和白细胞介素体外诱导培养小鼠脾脏细胞,可生成大量功能成熟的树突状细胞.     

Tuftsin stimulates IL-1 production by human mononuclear cells, human spleen cells and mouse spleen cells in vitro

Human peripheral adherent cells from splenectomized subjects, human spleen cells and mouse spleen cells were tested for IL-1 production in vitro in presence or absence of synthetic tuftsin (Thr-Lys-Pro-Arg). Application of synthetic tuftsin to peripheral blood adherent cells from normal donors as well as from splenectomized subjects induces IL-1 production. In splenectomized subjects the extent of induction was more evident than in controls. In human splenic cells tuftsin stimulates IL-1 production without KLH or LPS. In mouse spleen cells tuftsin alone did not stimulate the IL-1 secretion. However, addition of tuftsin to mouse spleen cells incubated with KLH augmented significantly the IL-1 secretion. As removal of the spleen leads to tuftsin deficiency, our present findings may perhaps explain the fulminant nature of the postplenectomy sepsis and some immune disturbances described in the postplenectomy state.     

Activation in vitro of rheumatoid synovial collagenase from cell cultures.

Rheumatoid synovial cells dissociated from matrix and adherent to culture dishes released a latent form of collagenase into culture medium. Previous studies have shown that the latent enzyme does not complex with alpha2-macroglobulin and binds to fibrillar substrate. We now show that serum-free culture medium of the synovial cells contains an inhibitor of collagenase as well as latent enzyme; the two were separated on a column of acrylamide/agarose. Latent collagenase (estimated mol wt 45,000-49,000) was transformed by trypsin to active collagenase of approximately equal to mol wt 33,000. When mixed with inhibitor the active enzyme formed an inactive complex again with approximately equal to mol wt 45,000-49,000. The inhibitor(s) itself was found in one major peak of mol wt 33,000-35,000 and several minor peaks eluting with lower apparent molecular weight. Mersalyl, an organic mercurial compound, effectively activated latent collagenase producing an active enzyme with approximately equal to mol wt 33,000. Bacterial collagenase did not activate latent enzyme. We suggest that latent rheumatoid synovial collagenase, as it is harvested from synovial cells in culture, is an enzyme-inhibitor complex.     

Heinz bodies induce clustering of band 3, glycophorin, and ankyrin in sickle cell erythrocytes.

In earlier model studies we demonstrated that artificially denatured hemoglobin binds to and clusters the protein, band 3, in the plane of the erythrocyte membrane. To determine whether denatured hemoglobin also clusters band 3 in vivo, we have compared the locations of denatured hemoglobin aggregates (Heinz bodies) with band 3 in sickle cells using phase contrast and immunofluorescence microscopy. We report that where Heinz bodies are found associated with the cytoplasmic surface of the membrane, clusters of band 3 are usually colocalized within the membrane. In contrast, normal erythrocyte membranes and regions of sickle cell membranes devoid of Heinz bodies display an uninterrupted staining of band 3. Similarly, ankyrin and glycophorin are periodically seen to aggregate at Heinz body sites, but the degree of colocalization is lower than for band 3. These data demonstrate that the binding of denatured hemoglobin to the membrane forces a redistribution of several major membrane components.     

Mechanisms of mouse spleen dendritic cell function in the generation of influenza-specific, cytolytic T lymphocytes.

We have evaluated the capacity of dendritic cells to function as antigen-presenting cells (APCs) for influenza and have examined their mechanism of action. Virus-pulsed dendritic cells were 100 times more efficient than bulk spleen cells in stimulating cytotoxic T lymphocyte (CTL) formation. The induction of CTLs required neither exogenous lymphokines nor APCs in the responding T cell population. Infectious virus entered dendritic cells through intracellular acidic vacuoles and directed the synthesis of several viral proteins. If ultraviolet (UV)-inactivated or bromelain-treated viruses were used, viral protein synthesis could not be detected, and there was poor induction of CTLs. This indicated that dendritic cells were not capable of processing noninfectious virus onto major histocompatibility complex (MHC) class I molecules. However, UV-inactivated and bromelain-treated viruses were presented efficiently to class II-restricted CD4+ T cells. The CD4+ T cells crossreacted with different strains of influenza and markedly amplified CTL formation. Cell lines that lacked MHC class II, and consequently the capacity to stimulate CD4+ T cells, failed to induce CTLs unless helper lymphokines were added. Similarly, dendritic cells pulsed with the MHC class I-restricted nucleoprotein 147-155 peptide were poor stimulators in the absence of exogenous helper factors. We conclude that the function of dendritic cells as APCs for the generation of virus-specific CTLs in vitro depends measurably upon: (a) charging class I molecules with peptides derived from endogenously synthesized viral antigens, and (b) stimulating a strong CD4+ helper T cell response.     

Spontaneous production of transforming growth factor-beta 2 by primary cultures of bronchial epithelial cells. Effects on cell behavior in vitro.

The ability of airway epithelial cells to produce transforming growth factor-beta (TGF-beta) may be an important mechanism for the control of growth, differentiation, and repair of the airway epithelium. To determine whether airway epithelial cells are capable of producing TGF-beta, we examined primary cultures of bovine bronchial epithelial cells. Using a bioassay, TGF-beta activity was detected readily in media conditioned by bovine bronchial epithelial cells. Neutralizing antisera to TGF-beta 1 and TGF-beta 2 were used to demonstrate that the majority of the activity was of the TGF-beta 2 isoform. Interestingly, some of the TGF-beta activity was present in the conditioned media as "active" TGF-beta, not requiring acid activation. The production of TGF-beta was variable, depending on cell density and the presence of retinoic acid. The presence of endogenously produced active TGF-beta in the culture media was shown to modulate the behavior of the cell cultures as evidenced by the effects of TGF-beta-neutralizing antisera on cell size and fibronectin production. Our results suggest that active TGF-beta produced by airway epithelial cells may function in an autocrine or paracrine manner to modulate epithelial cell behavior.     

Toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in primary cultures of mouse astrocytes.

The conversion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to its toxic 1-methyl-4-phenylpyridinium (MPP+) metabolite catalyzed by monoamine oxidase (MAO) type B is likely to occur within glial cells in the central nervous system. In this study, primary cultures of mouse astrocytes were used to assess the biochemical and toxic consequences of exposure to MPTP. MPTP caused a concentration-dependent loss of cell viability. This effect was probably due to the intracellular generation of MPP+, because cytotoxicity was prevented by preincubation of astrocytes in the presence of MAO inhibitors. After addition of 250 microM MPTP, loss of cell viability was preceded by an increased rate of glucose utilization and lactate accumulation, and by depletion of ATP. The ratio between the rates of lactate production (0.37 mM/hr) and glucose consumption (0.2 mM/hr) was 1.85, indicating that most of the glucose present in the medium was stoichiometrically converted to lactate via glycolysis. A remarkable correlation was found between ATP depletion and cytotoxicity caused by MPTP, and, when astrocytes were incubated in glucose-free medium, both ATP depletion and loss of viability occurred more rapidly. Finally, even after exposure for several days, astrocyte death could be prevented by washing MPTP from the incubation medium, suggesting that MPP(+)-induced mitochondrial damage may be reversible. We conclude that prolonged exposure of astrocytes to MPTP may result in loss of viability via the MAO-dependent generation of MPP+ and the ability of this toxic metabolite to impair mitochondrial function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Haplotype-specific suppression of antibody responses in vitro. II. Suppressor factor produced by T cells and T cell hybridomas from mice treated as neonates with semiallogeneic spleen cells

Culture supernatant fluids from spleen cells from C57BL/10 or BALB/c mice neonatally treated with semiallogeneic (B 10.D2 x B10)F1 cells to induce haplotype-specific suppressor T cells and restimulated with macrophages syngeneic at I-A with the allogeneic haplotype encountered as neonates contain a soluble factor capable of suppressing primary in vitro antibody responses of normal syngeneic spleen cells in a non- antigen-specific manner. This haplotype-specific suppressor factor, TsF- H, has also been recovered in culture fluids of a T cell hybridoma produced by fusion of the AKR thymoma BW5147 and the haplotype-specific suppressor T cells. TsF-H is inactivated by low pH (3.5) trypsin, for 30 min at 50 degrees C, and has a molecular weight in the range of 45,000 to 68,000. Studies with specific immunoabsorbents demonstrate the presence of determinants encoded by the I-A subregion of the haplotype of the T cell producing TsF-H but not I-J subregion or immunoglobulin constant-region determinants on the TsF-H. Suppression is restricted to primary in vitro antibody responses, and not secondary antibody, mixed lymphocyte, or cytotoxic lymphocyte responses by spleen cells syngeneic at the I-A subregion of H-2 with the T cell producing the factor. The properties and activities of TsF-H and the haplotype- specific suppressor T cell are compared and contrasted with antigen- specific and genetically restricted suppressor T cells and their factors.